The development of capillary electrophoresis (CE) into a viable analytical separation technique was not immediately undertaken following its theoretical conception. Rather, it initially became practiced out of necessity when other separation techniques, such as high performance liquid chromatography (HPLC), gas chromatography (GC), and conventional slab-gel electrophoresis failed to provide the resolution, efficiency, desired small sampling size, and/or reproducibility needed for analyzing increasingly complex mixtures of a wide variety of analytes. During the early developmental stages of CE, it became increasingly evident that conventional methods of chromatography were not living up to the standards needed for reproducibility for microscale analysis of certain biological samples. Although HPLC was proven to be successful for the analysis of oligonucleotides, peptides, and small proteins, poor resolution was often observed with large molecules such as DNA, RNA, and structural proteins. Although the technique was not immediately implemented, capillary electrophoresis has emerged as suitable technique for many types of samples and instruments can now be found in virtually every type of laboratory ranging from biological, environmental and even forensic.
Electrophoresis is defined as the movement of electrically charged ions in a condensed fluid due to an applied direct current electric field. This phenomenon was first observed in 1807 by Ferdinand Frederic Reuss, who noticed that the application of a constant electric field caused clay particles suspended in a water solution to migrate. He placed two vertical glass tubes in wet clay and filled the tubes with water while a layer of sand was placed in the bottom of each tube. The wires from a Voltaic Pile were inserted into the tubes and when the circuit was closed, the clay particles migrated through the layer of sand toward the positive pole, resulting in a cloudy solution. At the negative pole an increase in the volume of the solution was observed. The clay particles migrated because suspended particles in solution have a surface charge determined by the molecules absorbed on them. The surface of the clay particles had an excess of cations bonded to their surface, resulting in an excess positive charge and their net migration towards the cathode when the voltage was applied.
This principle also governs the separation mechanism of modern day capillary electrophoresis. The separation of analytes is primarily based on their different rates of migration (electrophoretic mobilities) in the bulk of the liquid phase. The rates of migration of the analytes are dependent on many factors, such as the strength of the applied electric field and viscosity of the solution, as well as each analyte’s hydrodynamic size-to-charge ratio. For a given charge and applied voltage, smaller analytes will travel faster than larger ones and will be detected sooner.
Capillary electrophoresis evolved from electrophoresis. The first electrophoresis experiment was pioneered by Tiselius. In his thesis titled, “The Moving Boundary Method to Study the Electrophoresis of Proteins” published in 1930, Tiselius utilized the electric charge carried by blood plasma proteins to achieve partial separations of proteins in free solution on a photographic film.In these studies, which contributed to him receiving the Nobel Prize in 1948, the instrumentation, which is shown in Figure 1, consisted of a U-shaped quartz cell filled with a buffer solution centered between two electrodes which were immersed at each end of the cell. When a voltage was applied, the compounds would migrate to the anode or cathode depending on their respective charges. Detection was achieved by monitoring the change in the refractive index at the boundary of the separated compounds using Schlieren optics at both ends of the solution in the cell [1]. Although refinements of this method were made spanning over more than two decades, widespread adaptation of the moving boundary electrophoresis technique was limited by its weaknesses: incomplete separation of the analytes of interest due to Joule heating problems and the large sample sizes required for analysis.
It is important to note that when current is passed through a resistive medium, which occurs when a voltage is applied, electrical heating, also known as Joule heating, is generated [2]. Heat is generated through the separation cell but dissipated only at the walls, giving rise to temperature gradients within the bulk liquid, which also gives rise to viscosity gradients. These phenomena translate into broadened peaks and decreased resolution. Peaks become broad because ions near the wall of the separation cell, where heat can be effectively dissipated, travel more slowly than the ions in the middle of the cell where the temperature is highest, and viscosity is lowest. To circumvent this problem, electrophoresis was carried out either with additional force fields [3] or a supporting medium such as paper, cellulose acetate, starch, agarose, and polyacrylamide gel [4-10]. The advent of using polyacrylamide gel was of importance because the mobility of the protein could be determined along with a rough estimate of its molecular weight. The use of filter paper as a supporting medium for electrophoresis has been widely used since the late 1940s. The use of an anti-convective supporting media allows analytes to become separated into "zones" which provide a more complete separation. However, these stabilizers may introduce other undesired effects such as the analyte binding to the stabilizer or sieving through the gel matrix, both conditions which would lead to analytes not be detected or eluted from the column [11].
To overcome the problems mentioned above regarding anti-convective stabilizers, Hjerten carried out electrophoresis using 1 to 3 mm internal diameter (i.d.) quartz tubes which rotated along its horizontal axis at approximately forty rotations per minute (rpm) in order to randomize the movement of ions in the tube and to help create a more even heating environment in the tube. Because no stabilizers were used, this technique became known as free zone electrophoresis. Some experiments also made use of a temperature controlled water bath in which the capillary tube was immersed to further reduce Joule heating effects. Although peak broadening from convection was essentially eliminated in this technique from the combination of the water bath and tube rotation, broadening from analyte adsorption to the wall of the tube was problematic, especially when dealing with proteins In these experiments, the internal surface of the tube was coated with methylcellulose in order to reduce electroosmotic flow and adsorption effects [12]. Analytes were detected by moving the tube back and forth across a fixed-position UV detector at the start of the experiment and then during regular intervals after the voltage was applied. While these experiments exemplified the power of free zone electrophoresis as a separation technique, few experiments were done in the 1970s because it did not draw enough attention from researchers. While the ideas and instrumentation of CE were available, but there was a culture of doing most analytical work by wet chemical methods. There was no particular urgency, financial or temporal, to analyze mixtures using what was deemed to be quite complicated instrumentation. However, methods that provide rapid means of characterization are essential tools in the scientific community and soon picked up interest when easier means of analysis were available.
In 1979, Mikkers et al. introduced free zone electrophoresis in small inner diameter Teflon tubes (0.2 mm i.d.) [11]. Small diameter tubes resulted in greatly reduced convection and current (μA vs. mA) as the temperature difference from the center of the tube to the wall is proportional to the square of the radius. In addition, smaller diameter tubes exhibit greatly enhanced heat dissipation because an ion will diffuse across a tube more often in a smaller diameter tube than a larger one. As a result of reducing Joule heat problems, higher voltages can be applied which provides faster analytes times. Voltages of up to 30,000 volts can be employed in some situations. Although this technique demonstrated the high efficiency characteristics of CE, the detection method used (conductometric) used was rather insensitive and thus the results did not attract much interest from the scientific community.
The real push toward current capillary electrophoresis experiments came in 1981 when Jorgenson and Lukacs demonstrated the use of even narrower capillaries (less than 100 mm inner diameter) to produce high efficiency (of more than 4×105 theoretical plates) for the separation of dansyl and fluorescamine derivatives of amino acids, dipeptides, and simple amines [13,14,15]. The number of theoretical plates in a chromatographic system is a measure of that system’s ability to maximize the separation of analytes while minimizing peak broadening. In contrast to previous CE experiments, where electroosmosis was suppressed, they took advantage of this new unique flow profile pioneered by Pretorius et al [16]. Unlike HPLC or GC separations, CE provided a flat flow profile instead of a parabolic flow profile, which helped to achieve the high resolution needed for the separation of complex mixtures. This flat profile originates because of where the electroosmotic flow is produced. Since the EOF originates near the wall of open capillary tubes, the bulk solution flows much more evenly through the capillary compared to conventional gas or liquid chromatography, which utilizes pressure driven flow. The high electroosmotic flow caused by the charge on the underivatized silica surface was caused the net migration of both anions and cations past the detection window. The CE systems were also much simpler than previous attempts, consisting of a fused silica capillary, electrode buffer reservoirs, a high voltage power supply and an optical detector from an HPLC. The detection window was made by simply burning a portion of the polyimide coating around the capillary. Samples were introduced by dipping the capillary inlet into the sample solution and applying voltage or raising the level of the sample vial. Although effective for the separation of charged ions, this technique was ineffective for the separation of neutral species.
This major limitation of CE was overcome in 1984 by Terabe, with the development of micellar electrokinetic chromatography (MEKC) [17]. In this technique, anionic micelles were utilized as a pseudostationary phase to separate neutral compounds. Sodium dodecyl sulfate surfactants were employed at a concentration greater than the critical micelle concentration. Above this concentration, surfactant monomers are at equilibrium with the micelles. Since they are of negative charge, the anionic micelles move counter to the electroosmotic flow, although their net migration is in the same direction as the electroosmotic flow. All other parameters of conventional CE methods were kept the same. Since the inception of MEKC, a variety of pseudostationary phases have been developed, including anionic, neutral, and cationic micelles and vesicles, as well as microemulsions, crown ethers, cyclodextrins and polymers. Unlike conventional zone electrophoresis, MEKC allowed the simultaneous separation of anionic, cationic, as well as neutral species.
After the introduction of commercial CE instrument in late 1988 that allowed the full automation of CE analysis to be possible, more and more research publications and industrial applications have made capillary electrophoresis be one of the dominant technologies in the separation field.
After the introduction of commercial CE instrument in late 1988 that allowed the full automation of CE analysis to be possible, more and more research publications and industrial applications have made capillary electrophoresis be one of the dominant technologies in the separation field. Figure 2 depicts capillary electrophoresis instrumentation today. The capillary tube is typically composed of fused silica with an inner diameter of 25-75 mm. Typical capillary lengths range from 30-100 cm depending on the analysis. The capillary tube, source and destination values are filled with buffer of a composition specific to the needs of the separation, which is used to maintain the source of electroosmotic flow. Samples are injected into the capillary and then the buffer is applied again. When a voltage is applied, the solutes migrate through the capillary as zones, depending on their electrophoretic mobilites. While the solutes are separating from each other due to their differences in their rates of migration, the strong electroosmotic flow moves the bulk solution through the capillary from the anode to the cathode. Cations travel slightly ahead of the EOF toward the detector located near the cathodic end of the capillary. Neutrals are not separated from one another but are pushed through the capillary by the EOF, except in the case of MEKC. Anions, which swim upstream against the EOF, require an EOF of high enough magnitude to carry the anions past the detector in order to simultaneously analyze anions and cations. If a positive voltage is applied, the elution order is cations, neutrals, and anions.
Figure 2. Diagram of capillary electrophoresis instrument [18].
One major application of CE has been for the analysis of DNA [19], which has been developed by a number of scientists including Kasper et al. 1998. Kasper hypothesized that the properties of fused silica capillaries and the variety of separation strategies that may be employed in capillary electrophoresis make it an ideal medium for electrophoresis of DNA in free solution [20]. They then designed separations of oligonucleotides and restriction fragments up to 23kb in size. Utilizing different CE methods, the authors were able to detect DNA over 3 orders of magnitude. They found that DNA stained with ethidium bromide dye shows greatest sensitivity at low DNA concentrations, contrary to traditional gel staining which shows a linear response over a wide range of DNA concentrations. The authors also tested the effectiveness of absorbance detection versus fluorescence detection, and found that absorbance detection of DNA is more selective and showed a larger linear response over a large range of concentrations, but stained DNA reduces background noise and eliminates certain artifacts, resulting in a lower limit of detection. The effect of the size and shape of the DNA molecules on the separation results were also investigated. The authors tested the resolution that could be achieved by CE of oligonucleotides which differ by a single base, and found that oligonucleotides that vary in size between 4 and 22 bases can be separated with simply the additional of 5% ethylene glycol. For the separation of larger DNA molecules, they found that the tertiary structure of the DNA molecule has a large effect on its electrophoretic mobility. Linear DNA has a much larger Stokes radius than tightly wound DNA, resulting in a difference in their viscous drag and therefore causing their separation. The highest resolution was achieved not by free zone electrophoresis or even capillary gel electrophoresis, but rather by specific complexation. The authors added a CTAB-micelle buffer to their standard procedure, which acted as an ion-pairing agent and greatly increased the resolution of the separation, resulting in a maximum efficiency for the system of ~1,000,000 theoretical plates. Compared with the normal efficiency of HPLC of ~20,000, their system increased the resolution achievable by more than 7-fold.
The general acceptance of capillary electrophoresis as a competitive separation technique required the convergence of three technologies: development of successful methods from conventional electrophoresis, development of high sensitivity optical detectors from HPLC, and the development of cheap fused silica tubing from gas chromatography. Although slow to widespread acceptance, capillary electrophoresis is currently one of the major chromatographic techniques and is sure to continue to play a leading role in the analysis of more and more complex biological samples and pharmaceuticals.
References
1. Tiselius, A., A new apparatus for electrophoretic analysis of colloidal mixtures. Trans. Faraday Soc.1937, 33, 524-531.DOI 2. Hinckley, J. O. N., Electrophoretic thermal theory. I. Temperature gradients and their effects. J. Chromatogr.1975, 109 (2), 209-17. DOI 3. Pfann, W. G.; Van Roosbroeck, W., Radioactive and photoelectric p-n junction power source. J. Appl. Phys.1954, 25, 1422-1434.DOI
4. Hjerten, S.; Jerstedt, S.; Tiselius, A., Electrophoretic particle sieving in polyacrylamide gels as applied to ribosomes. Anal. Biochem.1965, 11 (2), 218-218. DOI 5. Shapiro, A.; Vinuela, E.; Maizel Jr., J. V., Molecular weight estimation of polypeptide chains by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. Biochem. Biophys. Res. Commun.1967,28 (5), 815-20. DOI 6. Neville, D. M., Molecular weight determination of protein-dodecyl sulfate complexes by gel electrophoresis in a discontinuous buffer system. J. Biol. Chem. 1971, 246, 6328-6334. DOI 7. Rice, C. L.; Whitehead, R., Electrokinetic flow in a narrow cylindrical capillary. J. Phys. Chem.1965, 69 (11), 4017. DOI 8. Raymond, S.; Weintraub, L., Acrylamide gel as a supporting medium for zone electrophoresis. Science1959,130, 711. DOI 9. Brown, J. F.; Hinckley, J. O. N., Electrophoretic thermal theory II. Steady-state radial temperature gradients in circular section columns. J. Chromatogr.1975, 109, 218-224. DOI 10. Everaerts, F. M.; Hoving-Keulemans, W. M. L., Zone electrophoresis in capillary tubes. Sci. Tools1970, 17, 25-28. 11. Mikkers, F. E. P.; Everaerts, F. M.; Verheggen, T. P. E. M., High-performance zone electrophoresis J. Chromatogr.1979, 169 (1), 11-20. DOI 12. Hjerten, S., High-performance electrophoresis- Elimination of electroendosmosis and solute adsorption. J. Chromatogr.1985, 347 (2), 191-198.DOI 13. Jorgenson, J. W.; Lukacs, K. D., Zone electrophoresis in open-tubular glass capillaries. Anal. Chem. 1981, 53 (8), 1298-1302. DOI 14. Jorgenson, J.W.; Lukacs, K.D., High resolution separations based on electrophoresis and electroosmosis, J. Chromatogr.1987, 218, 209-216. DOI 15. Jorgenson, J.W.; Lukacs, K. D., Capillary zone electrophoresis. Science1983, 222, 266-272. DOI 16. Pretorius, V.; Hopkins, B. J.; Schieke, J. D., Electroosmosis: A new concept for high-speed liquid chromatography. J. Chromatogr. 1974, 99, 23-30. DOI 17. Terabe, S.; Otsuka, K.; Ichikawa, K.; Tsuchiya, A.; Ando, T., Electrokinetic separations with micellar solutions and open-tubular capillaries. Anal. Chem. 1984,56 (1), 111-113.DOI 18. Capillary Electrophoresis. http://en.wikipedia.org/wiki/Capillary_Electrophoresis (accessed 4 December 2012). Part of Wikipedia. Link 19. Drossman, H.; Luckey, J. A.; Kostichka, A.; D’Cunha, J.; Smith, L. M., High-speed separations of DNA sequencing reactions by capillary electrophoresis. Anal. Chem. 1990, 62 (9), 900-903. DOI 20. Kasper, T., et al. (1988). Separation and Detection of DNA by Capillary Electrophoresis. Journal of Chromatography. 458, 303-312 DOI Link
Development of Capillary Electrophoresis
The development of capillary electrophoresis (CE) into a viable analytical separation technique was not immediately undertaken following its theoretical conception. Rather, it initially became practiced out of necessity when other separation techniques, such as high performance liquid chromatography (HPLC), gas chromatography (GC), and conventional slab-gel electrophoresis failed to provide the resolution, efficiency, desired small sampling size, and/or reproducibility needed for analyzing increasingly complex mixtures of a wide variety of analytes. During the early developmental stages of CE, it became increasingly evident that conventional methods of chromatography were not living up to the standards needed for reproducibility for microscale analysis of certain biological samples. Although HPLC was proven to be successful for the analysis of oligonucleotides, peptides, and small proteins, poor resolution was often observed with large molecules such as DNA, RNA, and structural proteins. Although the technique was not immediately implemented, capillary electrophoresis has emerged as suitable technique for many types of samples and instruments can now be found in virtually every type of laboratory ranging from biological, environmental and even forensic.
Electrophoresis is defined as the movement of electrically charged ions in a condensed fluid due to an applied direct current electric field. This phenomenon was first observed in 1807 by Ferdinand Frederic Reuss, who noticed that the application of a constant electric field caused clay particles suspended in a water solution to migrate. He placed two vertical glass tubes in wet clay and filled the tubes with water while a layer of sand was placed in the bottom of each tube. The wires from a Voltaic Pile were inserted into the tubes and when the circuit was closed, the clay particles migrated through the layer of sand toward the positive pole, resulting in a cloudy solution. At the negative pole an increase in the volume of the solution was observed. The clay particles migrated because suspended particles in solution have a surface charge determined by the molecules absorbed on them. The surface of the clay particles had an excess of cations bonded to their surface, resulting in an excess positive charge and their net migration towards the cathode when the voltage was applied.
This principle also governs the separation mechanism of modern day capillary electrophoresis. The separation of analytes is primarily based on their different rates of migration (electrophoretic mobilities) in the bulk of the liquid phase. The rates of migration of the analytes are dependent on many factors, such as the strength of the applied electric field and viscosity of the solution, as well as each analyte’s hydrodynamic size-to-charge ratio. For a given charge and applied voltage, smaller analytes will travel faster than larger ones and will be detected sooner.
Capillary electrophoresis evolved from electrophoresis. The first electrophoresis experiment was pioneered by Tiselius. In his thesis titled, “The Moving Boundary Method to Study the Electrophoresis of Proteins” published in 1930, Tiselius utilized the electric charge carried by blood plasma proteins to achieve partial separations of proteins in free solution on a photographic film.In these studies, which contributed to him receiving the Nobel Prize in 1948, the instrumentation, which is shown in Figure 1, consisted of a U-shaped quartz cell filled with a buffer solution centered between two electrodes which were immersed at each end of the cell. When a voltage was applied, the compounds would migrate to the anode or cathode depending on their respective charges. Detection was achieved by monitoring the change in the refractive index at the boundary of the separated compounds using Schlieren optics at both ends of the solution in the cell [1]. Although refinements of this method were made spanning over more than two decades, widespread adaptation of the moving boundary electrophoresis technique was limited by its weaknesses: incomplete separation of the analytes of interest due to Joule heating problems and the large sample sizes required for analysis.
Figure 1. Moving Boundary Electrophoresis Instrumentation [1].
It is important to note that when current is passed through a resistive medium, which occurs when a voltage is applied, electrical heating, also known as Joule heating, is generated [2]. Heat is generated through the separation cell but dissipated only at the walls, giving rise to temperature gradients within the bulk liquid, which also gives rise to viscosity gradients. These phenomena translate into broadened peaks and decreased resolution. Peaks become broad because ions near the wall of the separation cell, where heat can be effectively dissipated, travel more slowly than the ions in the middle of the cell where the temperature is highest, and viscosity is lowest. To circumvent this problem, electrophoresis was carried out either with additional force fields [3] or a supporting medium such as paper, cellulose acetate, starch, agarose, and polyacrylamide gel [4-10]. The advent of using polyacrylamide gel was of importance because the mobility of the protein could be determined along with a rough estimate of its molecular weight. The use of filter paper as a supporting medium for electrophoresis has been widely used since the late 1940s. The use of an anti-convective supporting media allows analytes to become separated into "zones" which provide a more complete separation. However, these stabilizers may introduce other undesired effects such as the analyte binding to the stabilizer or sieving through the gel matrix, both conditions which would lead to analytes not be detected or eluted from the column [11].
To overcome the problems mentioned above regarding anti-convective stabilizers, Hjerten carried out electrophoresis using 1 to 3 mm internal diameter (i.d.) quartz tubes which rotated along its horizontal axis at approximately forty rotations per minute (rpm) in order to randomize the movement of ions in the tube and to help create a more even heating environment in the tube. Because no stabilizers were used, this technique became known as free zone electrophoresis. Some experiments also made use of a temperature controlled water bath in which the capillary tube was immersed to further reduce Joule heating effects. Although peak broadening from convection was essentially eliminated in this technique from the combination of the water bath and tube rotation, broadening from analyte adsorption to the wall of the tube was problematic, especially when dealing with proteins In these experiments, the internal surface of the tube was coated with methylcellulose in order to reduce electroosmotic flow and adsorption effects [12]. Analytes were detected by moving the tube back and forth across a fixed-position UV detector at the start of the experiment and then during regular intervals after the voltage was applied. While these experiments exemplified the power of free zone electrophoresis as a separation technique, few experiments were done in the 1970s because it did not draw enough attention from researchers. While the ideas and instrumentation of CE were available, but there was a culture of doing most analytical work by wet chemical methods. There was no particular urgency, financial or temporal, to analyze mixtures using what was deemed to be quite complicated instrumentation. However, methods that provide rapid means of characterization are essential tools in the scientific community and soon picked up interest when easier means of analysis were available.
In 1979, Mikkers et al. introduced free zone electrophoresis in small inner diameter Teflon tubes (0.2 mm i.d.) [11]. Small diameter tubes resulted in greatly reduced convection and current (μA vs. mA) as the temperature difference from the center of the tube to the wall is proportional to the square of the radius. In addition, smaller diameter tubes exhibit greatly enhanced heat dissipation because an ion will diffuse across a tube more often in a smaller diameter tube than a larger one. As a result of reducing Joule heat problems, higher voltages can be applied which provides faster analytes times. Voltages of up to 30,000 volts can be employed in some situations. Although this technique demonstrated the high efficiency characteristics of CE, the detection method used (conductometric) used was rather insensitive and thus the results did not attract much interest from the scientific community.
The real push toward current capillary electrophoresis experiments came in 1981 when Jorgenson and Lukacs demonstrated the use of even narrower capillaries (less than 100 mm inner diameter) to produce high efficiency (of more than 4×105 theoretical plates) for the separation of dansyl and fluorescamine derivatives of amino acids, dipeptides, and simple amines [13,14,15]. The number of theoretical plates in a chromatographic system is a measure of that system’s ability to maximize the separation of analytes while minimizing peak broadening. In contrast to previous CE experiments, where electroosmosis was suppressed, they took advantage of this new unique flow profile pioneered by Pretorius et al [16]. Unlike HPLC or GC separations, CE provided a flat flow profile instead of a parabolic flow profile, which helped to achieve the high resolution needed for the separation of complex mixtures. This flat profile originates because of where the electroosmotic flow is produced. Since the EOF originates near the wall of open capillary tubes, the bulk solution flows much more evenly through the capillary compared to conventional gas or liquid chromatography, which utilizes pressure driven flow. The high electroosmotic flow caused by the charge on the underivatized silica surface was caused the net migration of both anions and cations past the detection window. The CE systems were also much simpler than previous attempts, consisting of a fused silica capillary, electrode buffer reservoirs, a high voltage power supply and an optical detector from an HPLC. The detection window was made by simply burning a portion of the polyimide coating around the capillary. Samples were introduced by dipping the capillary inlet into the sample solution and applying voltage or raising the level of the sample vial. Although effective for the separation of charged ions, this technique was ineffective for the separation of neutral species.
This major limitation of CE was overcome in 1984 by Terabe, with the development of micellar electrokinetic chromatography (MEKC) [17]. In this technique, anionic micelles were utilized as a pseudostationary phase to separate neutral compounds. Sodium dodecyl sulfate surfactants were employed at a concentration greater than the critical micelle concentration. Above this concentration, surfactant monomers are at equilibrium with the micelles. Since they are of negative charge, the anionic micelles move counter to the electroosmotic flow, although their net migration is in the same direction as the electroosmotic flow. All other parameters of conventional CE methods were kept the same. Since the inception of MEKC, a variety of pseudostationary phases have been developed, including anionic, neutral, and cationic micelles and vesicles, as well as microemulsions, crown ethers, cyclodextrins and polymers. Unlike conventional zone electrophoresis, MEKC allowed the simultaneous separation of anionic, cationic, as well as neutral species.
After the introduction of commercial CE instrument in late 1988 that allowed the full automation of CE analysis to be possible, more and more research publications and industrial applications have made capillary electrophoresis be one of the dominant technologies in the separation field.
After the introduction of commercial CE instrument in late 1988 that allowed the full automation of CE analysis to be possible, more and more research publications and industrial applications have made capillary electrophoresis be one of the dominant technologies in the separation field. Figure 2 depicts capillary electrophoresis instrumentation today. The capillary tube is typically composed of fused silica with an inner diameter of 25-75 mm. Typical capillary lengths range from 30-100 cm depending on the analysis. The capillary tube, source and destination values are filled with buffer of a composition specific to the needs of the separation, which is used to maintain the source of electroosmotic flow. Samples are injected into the capillary and then the buffer is applied again. When a voltage is applied, the solutes migrate through the capillary as zones, depending on their electrophoretic mobilites. While the solutes are separating from each other due to their differences in their rates of migration, the strong electroosmotic flow moves the bulk solution through the capillary from the anode to the cathode. Cations travel slightly ahead of the EOF toward the detector located near the cathodic end of the capillary. Neutrals are not separated from one another but are pushed through the capillary by the EOF, except in the case of MEKC. Anions, which swim upstream against the EOF, require an EOF of high enough magnitude to carry the anions past the detector in order to simultaneously analyze anions and cations. If a positive voltage is applied, the elution order is cations, neutrals, and anions.
Figure 2. Diagram of capillary electrophoresis instrument [18].
One major application of CE has been for the analysis of DNA [19], which has been developed by a number of scientists including Kasper et al. 1998. Kasper hypothesized that the properties of fused silica capillaries and the variety of separation strategies that may be employed in capillary electrophoresis make it an ideal medium for electrophoresis of DNA in free solution [20]. They then designed separations of oligonucleotides and restriction fragments up to 23kb in size. Utilizing different CE methods, the authors were able to detect DNA over 3 orders of magnitude. They found that DNA stained with ethidium bromide dye shows greatest sensitivity at low DNA concentrations, contrary to traditional gel staining which shows a linear response over a wide range of DNA concentrations. The authors also tested the effectiveness of absorbance detection versus fluorescence detection, and found that absorbance detection of DNA is more selective and showed a larger linear response over a large range of concentrations, but stained DNA reduces background noise and eliminates certain artifacts, resulting in a lower limit of detection. The effect of the size and shape of the DNA molecules on the separation results were also investigated. The authors tested the resolution that could be achieved by CE of oligonucleotides which differ by a single base, and found that oligonucleotides that vary in size between 4 and 22 bases can be separated with simply the additional of 5% ethylene glycol. For the separation of larger DNA molecules, they found that the tertiary structure of the DNA molecule has a large effect on its electrophoretic mobility. Linear DNA has a much larger Stokes radius than tightly wound DNA, resulting in a difference in their viscous drag and therefore causing their separation. The highest resolution was achieved not by free zone electrophoresis or even capillary gel electrophoresis, but rather by specific complexation. The authors added a CTAB-micelle buffer to their standard procedure, which acted as an ion-pairing agent and greatly increased the resolution of the separation, resulting in a maximum efficiency for the system of ~1,000,000 theoretical plates. Compared with the normal efficiency of HPLC of ~20,000, their system increased the resolution achievable by more than 7-fold.
The general acceptance of capillary electrophoresis as a competitive separation technique required the convergence of three technologies: development of successful methods from conventional electrophoresis, development of high sensitivity optical detectors from HPLC, and the development of cheap fused silica tubing from gas chromatography. Although slow to widespread acceptance, capillary electrophoresis is currently one of the major chromatographic techniques and is sure to continue to play a leading role in the analysis of more and more complex biological samples and pharmaceuticals.
References
1. Tiselius, A., A new apparatus for electrophoretic analysis of colloidal mixtures. Trans. Faraday Soc. 1937, 33, 524-531. DOI
2. Hinckley, J. O. N., Electrophoretic thermal theory. I. Temperature gradients and their effects. J. Chromatogr. 1975, 109 (2), 209-17. DOI
3. Pfann, W. G.; Van Roosbroeck, W., Radioactive and photoelectric p-n junction power source. J. Appl. Phys. 1954, 25, 1422-1434. DOI
4. Hjerten, S.; Jerstedt, S.; Tiselius, A., Electrophoretic particle sieving in polyacrylamide gels as applied to ribosomes. Anal. Biochem. 1965, 11 (2), 218-218. DOI
5. Shapiro, A.; Vinuela, E.; Maizel Jr., J. V., Molecular weight estimation of polypeptide chains by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. Biochem. Biophys. Res. Commun. 1967, 28 (5), 815-20. DOI
6. Neville, D. M., Molecular weight determination of protein-dodecyl sulfate complexes by gel electrophoresis in a discontinuous buffer system. J. Biol. Chem. 1971, 246, 6328-6334. DOI
7. Rice, C. L.; Whitehead, R., Electrokinetic flow in a narrow cylindrical capillary. J. Phys. Chem. 1965, 69 (11), 4017. DOI
8. Raymond, S.; Weintraub, L., Acrylamide gel as a supporting medium for zone electrophoresis. Science 1959,130, 711. DOI
9. Brown, J. F.; Hinckley, J. O. N., Electrophoretic thermal theory II. Steady-state radial temperature gradients in circular section columns. J. Chromatogr. 1975, 109, 218-224. DOI
10. Everaerts, F. M.; Hoving-Keulemans, W. M. L., Zone electrophoresis in capillary tubes. Sci. Tools 1970, 17, 25-28.
11. Mikkers, F. E. P.; Everaerts, F. M.; Verheggen, T. P. E. M., High-performance zone electrophoresis J. Chromatogr. 1979, 169 (1), 11-20. DOI
12. Hjerten, S., High-performance electrophoresis- Elimination of electroendosmosis and solute adsorption. J. Chromatogr. 1985, 347 (2), 191-198. DOI
13. Jorgenson, J. W.; Lukacs, K. D., Zone electrophoresis in open-tubular glass capillaries. Anal. Chem. 1981, 53 (8), 1298-1302. DOI
14. Jorgenson, J.W.; Lukacs, K.D., High resolution separations based on electrophoresis and electroosmosis, J. Chromatogr. 1987, 218, 209-216. DOI
15. Jorgenson, J.W.; Lukacs, K. D., Capillary zone electrophoresis. Science 1983, 222, 266-272. DOI
16. Pretorius, V.; Hopkins, B. J.; Schieke, J. D., Electroosmosis: A new concept for high-speed liquid chromatography. J. Chromatogr. 1974, 99, 23-30. DOI
17. Terabe, S.; Otsuka, K.; Ichikawa, K.; Tsuchiya, A.; Ando, T., Electrokinetic separations with micellar solutions and open-tubular capillaries. Anal. Chem. 1984, 56 (1), 111-113. DOI
18. Capillary Electrophoresis. http://en.wikipedia.org/wiki/Capillary_Electrophoresis (accessed 4 December 2012). Part of Wikipedia. Link
19. Drossman, H.; Luckey, J. A.; Kostichka, A.; D’Cunha, J.; Smith, L. M., High-speed separations of DNA sequencing reactions by capillary electrophoresis. Anal. Chem. 1990, 62 (9), 900-903. DOI
20. Kasper, T., et al. (1988). Separation and Detection of DNA by Capillary Electrophoresis. Journal of Chromatography. 458, 303-312 DOI Link